Polypeptide

ABSTRACT

The invention provides an immunoglobulin G Fc region binding polypeptide, which polypeptide comprises an immunoglobulin G Fc region binding motif, BM, consisting of an amino acid sequence selected from: i) EQQX 4 AFYEIL HLPNL-TEX 18 QX 20 X 21 AFIX 25 X 26 LRX 29 , and ii) an amino acid sequence which has at least 85% identity to the sequence defined in i). Also provided are methods of isolation or production of IgG Fc-containing molecules.

FIELD OF THE INVENTION

This invention relates to a polypeptide which binds to immunoglobulin GFc (IgG Fc). The polypeptide has industrial application for example inaffinity separation and/or purification in the production of antibodiesand/or Fc fusion proteins.

BACKGROUND

In the industrial production of monoclonal antibodies and Fc fusionproteins, purification is frequently carried out using affinitychromatography. Protein A from Staphylococcus aureus has long been usedas affinity ligand in such applications, due to the native affinity ofProtein A for the Fc portion of IgG. Protein A in its entirety, as wellas the individual Fc-binding domains thereof, have subsequently servedas starting points for the rational design of engineered affinityligands with improved properties. Despite the comparable success ofcurrently used IgG Fc affinity ligands, there is a continued need forimprovement. The continued provision of agents having an affinity forIgG Fc that is comparable with, or higher than, that exhibited byProtein A remains a matter of substantial interest. For example, ProteinA affinity chromatography typically uses low pH conditions, which maylead to loss of yield due to the sensitivity of several antibodies andFc fusion proteins to low pH conditions. The provision of new IgGFc-binding agents that allow elution at a higher pH as compared toProtein A during affinity chromatography would therefore be beneficial.

It is an object of the invention to provide new IgG Fc-binding agents,that could for example be used in the production of antibodies or Fcfusion proteins, e.g. for affinity separation and/or purification.

SUMMARY OF THE INVENTION

According to one aspect thereof, the invention provides animmunoglobulin G Fc (IgG Fc) binding polypeptide, comprising an IgGFc-binding motif, BM, which motif consists of an amino acid sequenceselected from:

i) EQQX₄AFYEIL HLPNLTEX₁₈QX₂₀ X₂₁AFIX₂₅X₂₆LRX₂₉,

-   -   wherein, independently of each other,    -   X₄ is selected from H and N;    -   X₁₈ is selected from D and G;    -   X₂₀ is selected from R and K;    -   X₂₁ is selected from H and Q;    -   X₂₅ is selected from R, A and G;    -   X₂₆ is selected from A, S and T; and    -   X₂₉ is selected from G, K and A;    -   and

-   ii) an amino acid sequence which has at least 85% identity to the    sequence defined in i).

The above definition of a class of sequence related, IgG Fc-bindingpolypeptides according to the invention is based on an analysis of anumber of random polypeptide variants of a parent scaffold, that wereselected from a combinatorial protein library for their interaction withIgG Fc in phage display selection experiments (Examples 1 and 2). Theidentified IgG Fc-binding motif, or “BM”, corresponds to the targetbinding region of the parent scaffold, which region constitutes twoalpha helices within a three-helical bundle protein domain. In theparent scaffold, the varied amino acid residues of the two BM helicesinclude amino acid residues that participate in the binding surface forinteraction with Fc. In the present invention, the random variation ofsurface residues and the subsequent selection of variants have modifiedthe original Fc interaction capacity.

As the skilled person will realize, the function of any polypeptide,such as the IgG Fc-binding capacity of the polypeptides according to theinvention, is dependent on the tertiary structure of the polypeptide. Itis therefore possible to make minor changes to the sequence of aminoacids in a polypeptide without affecting the function thereof. Thus, theinvention encompasses modified variants of the BM of i), which are suchthat the resulting sequence is at least 85% identical to a sequencebelonging to the class defined by i). For example, it is possible thatan amino acid residue belonging to a certain functional grouping ofamino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could beexchanged for another amino acid residue from the same functional group.

In one embodiment of the polypeptide according to the invention, X₄ isH.

In one embodiment of the polypeptide according to the invention, X₁₈ isG.

In one embodiment of the polypeptide according to the invention, X₂₀ isK.

In one embodiment of the polypeptide according to the invention, X₂₁ isH.

In one embodiment of the polypeptide according to the invention, X₂₅ isR.

In one embodiment of the polypeptide according to the invention, X₂₆ isA.

In one embodiment of the polypeptide according to the invention, X₂₉ isG.

As described in detail in the experimental section to follow, theselection of IgG Fc-binding variants has led to the identification ofindividual IgG Fc-binding motif (BM) sequences. These sequencesconstitute individual embodiments of the BM sequence i) in thedefinition of IgG Fc-binding polypeptides according to this aspect ofthe present invention. The sequences of individual IgG Fc-binding motifsare presented in FIG. 1 and as SEQ ID NO:1-3 (FIG. 1). In embodiments ofthis aspect of the invention, the BM sequence i) may in particular beSEQ ID NO:1.

In embodiments of the present invention, the BM may form part of athree-helix bundle protein domain. For example, the BM may essentiallyconstitute or form part of two alpha helices with an interconnectingloop, within said three-helix bundle protein domain.

In particular embodiments of the invention, such a three-helix bundleprotein domain is selected from domains of bacterial receptor proteins.Non-limiting examples of such domains are the five differentthree-helical domains of protein A from Staphylococcus aureus, andderivatives thereof. Thus, an IgG Fc-binding polypeptide according tothe invention may comprise an amino acid sequence selected from:

ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK(BM within domain A of staphylococcal protein A);ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK(BM within domain B of staphylococcal protein A);ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK(BM within domain C of staphylococcal protein A);ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK(BM within domain D of staphylococcal protein A);AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK(BM within domain E of staphylococcal protein A); andVDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK(BM within the protein Z derivative of domain Bof staphylococcal protein A);

wherein [BM] is an IgG Fc-binding motif as defined above.

In alternative embodiments of the present invention, wherein the BMagain essentially constitutes or forms part of two alpha helices with aninterconnecting loop, within said three-helix bundle protein domain, theIgG Fc-binding polypeptide comprises the amino acid sequence:

FWK-[BM]-DPSQSARLLAX_(a)AKKLDDQ,wherein [BM] is an IgG Fc-binding motif as defined above, and X_(a) isselected from R, G and Q.

For example, the IgG Fc-binding polypeptide may comprise the amino acidsequence:

VDAKFWK-[BM]-DPSQSARLLAX_(a)AKKLDDQAPKwherein [BM] is an IgG Fc-binding motif as defined above, and X_(a) isselected from R, G and Q.

In some examples of these embodiments, X_(a) is R.

The IgG Fc-binding polypeptide may for example comprise an amino acidsequence selected from SEQ ID NO:4-6, such as SEQ ID NO:4 (FIG. 1).

According to another alternative aspect thereof, the invention providesan IgG Fc-binding polypeptide, whose amino acid sequence comprises asequence which fulfils one definition selected from the following: iii)it is selected from SEQ ID NO:7-9, and iv) it is an amino acid sequencehaving 85% or greater identity to a sequence selected from SEQ ID NO:7-9(FIG. 1). In embodiments of this aspect of the invention, the IgGFc-binding polypeptide may in particular comprise SEQ ID NO:7, or asequence having 85% or greater identity thereto.

When reference is made herein to the degree of identity between theamino acid sequences of different polypeptides, the lower limit of 85identity to a sequence disclosed herein is given. In some embodiments,the inventive polypeptide may have a sequence which is at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to the sequencedescribed herein. The comparison may be performed over a windowcorresponding to the shortest of the sequences being compared, or over awindow corresponding to an IgG Fc-binding motif in at least one of thesequences being compared.

An IgG Fc-binding polypeptide according to any aspect of the inventionmay bind to IgG Fc such that the K_(D) value of the interaction is atmost 1×10⁻⁶ M, for example at most 1×10⁻⁷ M, such as at most 5×10⁻⁸ M.

The polypeptide is advantageous in that it binds well to an IgG Fc. Inparticular, the polypeptide may be capable of binding to the Fc portionof a human IgG molecule. In some embodiments of the invention, thepolypeptide is capable of binding to classes 1, 2 and 4 of human IgG,but not to class 3. In some embodiments, the polypeptide is capable ofbinding to the interface between the CH2 and CH3 domains of IgG Fc. Insome embodiments, the polypeptide is capable of binding to an area onthe Fc molecular surface made up by the Fc amino acid residuesT250-S254, T256, L309-L312, L314, D315, E430 and L432-Y436 (numberingaccording to Deisenhofer, Biochemistry (1981) 20(9):2361-70).

The skilled addressee will appreciate that various modifications and/oradditions can be made to a polypeptide according to the invention inorder to tailor the polypeptide to a specific application withoutdeparting from the scope of the present invention. These modificationsand additions are described in more detail below and may includeadditional amino acids in the same polypeptide chain, or labels and/ortherapeutic agents that may be chemically conjugated or otherwise boundto the polypeptide of the invention.

Furthermore, the invention also encompasses fragments of IgG Fc-bindingpolypeptides according to the invention that retain IgG Fc-binding. Thepossibility of creating fragments of a wild-type Staphylococcus aureusprotein A domain with retained binding specificity was shown by BraistedA C et al in Proc Natl Acad Sci USA 93:5688-5692 (1996). In theexperiments described in that paper, using a structure-based design andphage display methods, the binding domain of a three-helix bundle of 59residues was reduced to a resulting two-helix derivative of 33 residues.This was achieved by stepwise selection of random mutations fromdifferent regions, which caused the stability and binding affinity to beiteratively improved. Following the same reasoning, with thepolypeptides of the present invention, the skilled addressee will beable to obtain a “minimized” IgG Fc-binding polypeptide with the samebinding properties as that of the “parent” IgG Fc-binding polypeptide.Thus, a polypeptide constituting a fragment of a polypeptide accordingto the invention is within the scope of the invention.

The terms “IgG Fc-binding” and “binding affinity for IgG Fc” as used inthis specification refers to a property of a polypeptide which may betested for example by the use of surface plasmon resonance technology,such as in a Biacore instrument (GE Healthcare). For example asdescribed in the examples below, IgG Fc-binding affinity may be testedin an experiment in which IgG Fc, or a fragment of IgG Fc, isimmobilized on a sensor chip of the instrument, and the samplecontaining the polypeptide to be tested is passed over the chip.Alternatively, the polypeptide to be tested is immobilized on a sensorchip of the instrument, and a sample containing IgG Fc, or fragmentthereof, is passed over the chip. The skilled person may then interpretthe results obtained by such experiments to establish at least aqualitative measure of the binding affinity of the polypeptide for IgGFc. If a quantitative measure is desired, for example to determine aK_(D) value for the interaction, surface plasmon resonance methods mayalso be used. Binding values may for example be defined in a Biacore2000 instrument (GE Healthcare). IgG Fc is immobilized on a sensor chipof the measurement, and samples of the polypeptide whose affinity is tobe determined are prepared by serial dilution and injected in randomorder. K_(D) values may then be calculated from the results using forexample the 1:1 Langmuir binding model of the BIAevaluation 4.1 softwareprovided by the instrument manufacturer.

Where amino acid substitutions are introduced, these should not affectthe basic structure of the polypeptide. For example, the overall foldingof the Cα backbone of the polypeptide can be essentially the same asthat of a domain of protein A, i.e. having the same elements ofsecondary structure in the same order. Thus, polypeptides having thisbasic structure will have similar CD spectra to the wild-type protein Adomain. The skilled addressee is aware of other parameters that may berelevant. The requirement of conserving the basic structure, placesrestrictions on which positions of the amino acid sequence may besubject to substitution. For example, it is preferred that amino acidresidues located on the surface of the polypeptide are substituted,whereas amino acid residues buried within the core of the polypeptide“three-helix bundle” should be kept constant in order to preserve thestructural properties of the molecule. The same reasoning applies tofragments of polypeptides of the invention.

The invention also covers polypeptides in which the IgG Fc-bindingpolypeptide described above is present as an IgG Fc-binding domain towhich additional amino acid residues have been added at either terminal.These additional amino acid residues may play a role in the binding ofIgG Fc by the polypeptide, but may equally well serve other purposes,related for example to one or more of the production, purification,stabilization in vivo and/or in vitro, coupling or detection of thepolypeptide. Such additional amino acid residues may comprise one ormore amino acid residues added for the purpose of chemical coupling. Oneexample of this is the addition of a cysteine residue N-terminally orC-terminally with respect to the binding motif, e.g. close to or at theN or C terminus. Such additional amino acid residues may also provide a“tag” for purification or detection of the polypeptide such as a His₆tag or a “myc” (c-myc) tag or a “FLAG” tag for interaction withantibodies specific to the tag.

The present invention also covers IgG Fc-binding polypeptides in whichan IgG Fc-binding polypeptide as described above is present as an IgGFc-binding domain to which additional peptides or proteins or otherfunctional groups are coupled N- or C-terminally or to any otherresidues (specifically or non-specifically) by means of chemicalconjugation (using known organic chemistry methods).

The “additional amino acid residues” discussed above may also provideone or more polypeptide domains with any desired function, such as thesame binding function as the first, IgG Fc-binding domain, or anotherbinding function, or an enzymatic function, toxic function (e.g. animmunotoxin), or a fluorescent signaling function, or combinationsthereof.

The polypeptide of the invention may be in monomeric or multimericforms. Multimeric forms of the polypeptide may be advantageous in thatthey may have enhanced binding properties. Preferred multimeric formsinclude dimeric, trimeric and tetrameric forms. Multimeric forms of thepolypeptides may comprise a suitable number of polypeptides of theinvention. These polypeptides essentially form domains within themultimer. These domains may all have the same amino acid sequence, butalternatively, they may have different amino acid sequences. Thepolypeptides may be joined by covalent coupling using known organicchemistry methods, or expressed as one or more fusion polypeptides in asystem for recombinant expression of polypeptides, or joined in anyother fashion, either directly or via a linker, for example an aminoacid linker.

Additionally, fusion polypeptides, in which the IgG Fc-bindingpolypeptide of the invention provides a first domain or moiety, andsecond or further moieties have other functions than binding IgG Fc arealso contemplated and within the scope of the present invention. Thesecond or further moieties of such a fusion polypeptide may comprise abinding domain with an affinity for another target molecule than IgG Fc.Such a binding domain may be another, similar polypeptide binder. Forexample, the polypeptide binder may be a variant of protein Z derivedfrom domain B of protein A. This makes it possible to createmulti-specific reagents that may be used in several types ofapplications such as medicine, veterinary medicine, diagnosis,separation, and imaging. The preparation of such multi-specific fusionpolypeptides may be performed using methods well known in the art ofmolecular biology.

In other embodiments of the invention, the second or further moietiesmay comprise an unrelated, naturally occurring or recombinant protein(or a fragment thereof which retains the binding or other ability of thenaturally-occurring or recombinant protein) having a binding affinityfor a target. For example, an IgG Fc-binding polypeptide in accordancewith the invention may be joined to an albumin-binding domain, such asthe albumin binding domain GA3 of protein G from Streptococcus strainG148 (“ABD”), or any other polypeptide with affinity for a serumprotein.

The IgG Fc-binding polypeptides of the present invention may be providedin the form of other fusion polypeptides. For example the IgG Fc-bindingpolypeptide, or fragment thereof, may be covalently coupled to a secondor further moiety or moieties, which in addition to, or instead oftarget binding, exhibit other functions. One example would be a fusionbetween one or more IgG Fc-binding polypeptides and an enzymaticallyactive polypeptide serving as a reporter or effector moiety. Examples ofreporter enzymes, which may be coupled to the IgG Fc-binding polypeptideto form a fusion protein, are well-known to the skilled person andinclude enzymes such as β-galactosidase, alkaline phosphatase,horseradish peroxidase, carboxypeptidase. Other options for the secondand further moiety or moieties of a fusion polypeptide according to theinvention include fluorescent polypeptides, such as green fluorescentprotein, red fluorescent protein, luciferase and variants thereof.

A polypeptide according to the invention may be useful in any methodwhich relies on affinity for IgG Fc of a reagent. Thus, the polypeptidemay be used as a detection reagent, a capture reagent or a separationreagent in such methods. In particular, the polypeptide exhibits severalcharacteristics which make it useful as an affinity reagent in affinitychromatography, wherein the goal is to separate, purify and/or produceantibodies or Fc fusion proteins from a heterogeneous mixture. Thepolypeptide can be bound to a matrix and e.g. used for the purificationof IgG Fc-containing therapeutic compounds in industrial production. Dueto properties such as a high target affinity, a high stability both inacidic and basic environments and a high selectivity for the IgG Fcfragment over the IgG Fab fragment, the IgG Fc-binding polypeptideaccording to the invention is thought to present a very attractiveaffinity reagent.

Thus, another aspect of the present invention is a method of isolatingmolecules comprising IgG Fc from a sample, which method comprises thesteps:

(i) providing a sample containing molecules comprising IgG Fc;

(ii) contacting the sample with an IgG Fc-binding polypeptide asdescribed herein, whereby said molecules comprising IgG Fc bind to thepolypeptide;

(iii) isolating bound molecules comprising IgG Fc from the sample.

In the inventive isolation method, the sample may be derived from aculture of prokaryotic or eukaryotic, such as mammalian or plant, cellsexpressing molecules comprising IgG Fc, or from expression of suchmolecules in an alternative expression system, for example a vesicularsystem. Alternatively, the sample may be derived from transgenicexpression in a host, such as a plant or mammalian host.

In some embodiments, said molecules comprising IgG Fc are IgG moleculesor fragments thereof. For example, they can be human IgG molecules orfragments thereof. In some embodiments, said molecules comprising IgG Fcare monoclonal IgG antibodies. In particular, such monoclonal IgGantibodies may be human monoclonal IgG antibodies. For example, they arehuman monoclonal IgG antibodies from class 1, 2 and/or 4.

In other embodiments, said molecules comprising IgG Fc are Fc fusionproteins. The Fc domain in such a fusion protein may thus,advantageously, be used as an “affinity handle” in the isolation of thefusion protein. A large variety of Fc fusion proteins have been created.For example, Fc fusion proteins having therapeutic applications includeetanercept, which is a fusion between soluble TNF-α receptor and Fc, andVEGF Trap, which is a fusion between VEGF receptor domains and Fc(Holash et al, Proc Natl Acad Sci USA (2002) 99(17):11393-11398). Whilethese two are illustrative examples of great interest, the listing ofthem is non-limiting, and it is in principle possible to fuse an Fcdomain to any desired protein in order to modify its properties andfacilitate affinity purification thereof using the inventive IgGFc-binding polypeptide described herein as affinity ligand.

Yet another aspect of the present invention concerns a method ofproducing molecules comprising IgG Fc, which method comprises the steps:

(i) expressing desired molecules comprising IgG Fc;

(ii) obtaining a sample of molecules comprising IgG Fc from saidexpression;

(iii) contacting the sample with an IgG Fc-binding polypeptide asdescribed herein, whereby molecules comprising IgG Fc bind to thepolypeptide;

(iv) isolating bound molecules comprising IgG Fc from the sample, and

(v) recovering bound molecules comprising IgG Fc through elution thereoffrom the IgG Fc-binding polypeptide.

Expression step (i) may be performed using any known expression system,for example recombinant expression in prokaryotic or eukaryotic, such asmammalian or plant, cells, or in a vesicular system. The sample may alsobe derived from transgenic expression in a host, such as a plant ormammalian host.

In some embodiments, said molecules comprising IgG Fc are IgG moleculesor fragments thereof. For example, they can be human IgG molecules orfragments thereof. In some embodiments, said molecules comprising IgG Fcare monoclonal IgG antibodies. In particular, such monoclonal IgGantibodies may be human monoclonal IgG antibodies. For example, they arehuman monoclonal IgG antibodies from class 1, 2 and/or 4.

In other embodiments, said molecules comprising IgG Fc are Fc fusionproteins.

In some embodiments of the inventive methods of isolating and producing,the IgG Fc-binding polypeptide is immobilized on a chromatographymedium. In general, methods that employ the polypeptides in accordancewith the invention in vitro may be performed in different formats, suchas on filters or membranes, microtitre plates, in protein arrays, onbiosensor surfaces, on beads, in flow cytometry, on tissue sections, andso on. In a specific aspect, the invention provides an affinitychromatography medium, which has an IgG Fc-binding polypeptide asdescribed herein immobilized thereon. Such a medium may be based on anyknown chromatography material as a matrix, and coupling of thepolypeptide to the matrix may be performed using any one of severalknown procedures.

The numbering of amino acid residues and any use of the term “position”in the sequence of the polypeptide according to the invention isrelative. In a polypeptide in accordance with the invention which has asmany amino acid residues as a specifically disclosed polypeptide, i.e.those described above, the positions of amino acids in the polypeptidecorrespond exactly to those in the disclosed polypeptides. In asituation where there is, for example, an N terminal extension comparedto the disclosed polypeptides, those amino acid residues in the extendedpeptide that correspond to those of the non-extended peptide have thesame position numbers. For example, if there is a six amino acid residueextension on the extended polypeptide, then amino acid number seven ofthat modified polypeptide, counting from the N terminus, corresponds tothe amino acid in position number one of the disclosed polypeptide.

With regard to the description above of fusion polypeptides and proteinsincorporating an IgG Fc-binding polypeptide of the invention, it shouldbe noted that the designation of first, second and further moieties ismade for the purposes of clarity to distinguish between the IgGFc-binding moiety or moieties on the one hand, and moieties exhibitingother functions on the other hand. These designations are not intendedto refer to the actual order of the different domains in the polypeptidechain of the fusion protein or polypeptide. Thus, for example, a firstmoiety may appear at the N-terminal end, in the middle, or at theC-terminal end of the fusion protein or polypeptide.

The invention is further illustrated by the following non-limitingexamples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a listing of the amino acid sequences of examples of IgGFc-binding motifs comprised in IgG Fc-binding polypeptides of theinvention (SEQ ID NO:1-3), examples of IgG Fc-binding polypeptidesaccording to the invention (SEQ ID NO:4-9), the protein Z derivative ofdomain B of Staphylococcus aureus protein A (SEQ ID NO:10), and the Zvariant Z01730 previously obtained by phage display selection from acombinatorial protein library (SEQ ID NO:11).

FIG. 2 shows overlay of CD spectra taken at 195-250 nm before and aftervariable temperature measurement (VTM) involving heating at 90° C. forA: Z02829, B: Z02726 and C: Z02742. Z02829 is a derivative of Z02674containing two substitutions at the beginning of the protein (A8N andW11N with respect to the sequence of the entire expressed molecule, i.e.A3N and W6N with respect to SEQ ID NO:7). Thus, the IgG Fc-binding motifof Z02829 is the same as that of Z02674.

FIG. 3 shows sensorgrams obtained from Biacore analysis of IgGFc-binding molecules according to the invention. Sensorgrams obtainedafter injection of 25 nM of Z02674 (solid line), Z02726 (dashed line) orZ02742 (dotted line) over CM5 sensor-chip surfaces containingimmobilized palivizumab (A; 1280 response units, RU); trastuzumab (B;1200 RU) and etanercept (C; 1500 RU). Signal from a blank sensor-chipsurface was subtracted.

FIG. 4 shows sensorgrams obtained from kinetic Biacore analysis of IgGFc-binding molecules according to the invention. The overlay plots showsensorgrams obtained after injections over immobilized palivizumab of 25nM or 100 nM of Z02674 (A); Z02726 (B) and Z02742 (C) (dotted lines).The response curves were fitted to a 1:1 binding model (solid lines).

FIG. 5 shows chromatograms for columns comprising immobilized IgGFc-binding polypeptides according to the invention. The overlaychromatograms show elution profiles for etanercept (A), trastuzumab (B)and palivizumab (C) when eluted with an acidic pH gradient from columnscomprising Z00000, Z02742, Z02674 or Z02726, as indicated.

FIG. 6 shows a histogram of dynamic binding capacities (moles ofIgG/moles of polypeptide) for columns comprising immobilized Z00000,Z02742, Z02674 or Z02726, as indicated.

EXAMPLE 1 Selection and Initial Characterization of IgG Fc-BindingPolypeptides

In this Example, a Z variant library denoted ZLib2007-IgG, createdfollowing an initial selection of IgG binding Z variants from thelibrary ZLib2002 and evaluation of results, was used for selection ofIgG Fc-binding polypeptides according to the invention. Details oflibrary construction and selection procedures were generally asdescribed in Grönwall et al, J Biotechnol 128:162-183, 2007. Fourdifferent phage display selections from ZLib2007-IgG were made againstvarious IgG and IgG-like molecules. Clones were sequenced, the sequencesanalyzed by clustering and the amino acid sequence of each clonecompared to the distribution of variable amino acids among all selectedclones and the distribution in the library design. IgG Fc-bindingmolecules were chosen for further characterization after four selectionrounds, whereupon a fifth round was carried out with more stringentwashing conditions, elution at a higher pH and with a higher temperatureduring selection. Additional IgG Fc-binding molecules derived fromselection round 5 were selected for further characterization.

Materials and Methods Selection

Five selection rounds were performed in each one of four selectionsetups, and new phage stocks were prepared between each round. Targetprotein was alternated between selection rounds in some selectionsetups. Targets used were human immunoglobulin G2κ (IgG2κ) from myelomaserum (Meridian Life Science, cat. no. A50184H), human immunoglobulinG3λ (IgG3λ) from myeloma serum (Meridian Life Science, cat. no.A50186H), human immunoglobulin G4λ (IgG4λ) from myeloma serum (MeridianLife Science, cat. no. A50947H), etanercept (trade name Enbrel®;Apoteket cat. no. 566661, producer Wyeth, lot 21032), biotinylated humanimmunoglobulin G, Fc fragment (IgG-Fc) (Jackson Immunoresearch, cat. no.009-060-008, lot 66321), and biotinylated human immunoglobulin G1κ(IgG1κ) (Ancell, cat. no. 295-030, lot 141605). An overview of targetproteins in each round of each selection setup is presented in Table 1.The selections were performed against biotinylated target protein inliquid phase for selection against IgG variants, or against targetimmobilized on a solid phase in the form of the surface of an immunotubefor selection against etanercept.

TABLE 1 Selection setups Selection No. of Time/wash Elution Setup roundTarget Concentration washes (min) pH IgG_22-Sel1 1 Poly-IgG-Fc 100 nM 20 3.5 2 Etanercept 6 μg/ml 2 0 3.5 3 Poly-IgG-Fc 20 nM 4 1 3.5 4Etanercept 3 μg/ml 5 2 3.5 5 Poly-IgG-Fc 20 nM 9 5 3.8 IgG_21-Sel1 1Poly-IgG1&2&4 33 + 33 + 33 nM 2 0 3.5 2 Poly-IgG1&2&4 17 + 17 + 17 nM 20 3.5 3 Poly-IgG1&2&4 7 + 7 + 7 nM 4 1 3.5 4 Poly-IgG1&2&4 7 + 7 + 7 nM5 2 3.5 5 Poly-IgG1&2&4 7 + 7 + 7 nM 9 5 3.8/4.5 IgG_23-Sel1 1 Poly-IgG1100 nM 2 0 2.2 2 Poly-IgG4 50 nM 2 0 2.2 3 Poly-IgG2 20 nM 5 2 2.2 4Poly-IgG2 20 nM 7 3 2.2 5 Poly-IgG1 20 nM 13 7 3.8 IgG_21-Sel2 1Poly-IgG1&2&4 20 + 20 + 20 nM 2 0 2.2 2 Poly-IgG1&2&4 8 + 8 + 8 nM 2 02.2 3 Poly-IgG1&2&4 4 + 4 + 4 nM 4 1 2.2 4 Poly-IgG1&2&4 2 + 2 + 2 nM 52 2.2 5 Poly-IgG1&2&4 1 + 1 + 1 nM 9 5 3.8

Warm conditions for selection (37° C.) and wash (37-45° C.) were used inround 5 in all setups.

Phage library stock was PEG/NaCl precipitated twice and dissolved in 1ml selection buffer (PBS: 2.68 mM KCl, 1.47 mM KH₂PO₄, 137 mM NaCl, 8.1mM Na₂HPO₄, pH 7.4; supplemented with 0.1% Tween20 (Acros Organics cat.no. 2333 62500) and 0.1% gelatine (Prolabo, cat. no. 24 360.233)).

Liquid phase selection: Phages were pre-incubated with streptavidincoated beads (Dynabeads® M-280; Dynal cat. no. 112.06) for 1 hour atroom temperature. Pre-clearing against Fab was made in a Maxisorpimmunotube (Nunc, cat. no. 444202) coated with Fab. All tubes and beadsused in the selection procedure were pre-blocked in selection buffer.Phages were incubated with biotinylated target under agitation for up to3 hours. Then, the phages were transferred to pre-blocked streptavidinbeads and incubated for 15 min with agitation, and the beads were washedin selection buffer according to Table 1.

Solid phase selection: Target protein was immobilized onto immunotubes.Phages were pre-incubated in an immunotube coated with Fab. All tubes,including tubes coated with target, were blocked in selection bufferprior to selection. Phages were incubated with the immobilized targetmolecules under agitation and the tube was thereafter washed inselection buffer.

Elution and infection: Phages from either solid or liquid phaseselection were eluted with elution buffer (0.05 M glycine-HCl at pH 2.2,or 0.05 M NaAc buffer at pH 3.5, 3.8 or 4.5 as outlined in Table 1),followed by immediate neutralization with neutralization buffer (1 MTris-HCl, pH 8.0). The eluted phages (95% of the volume) were used toinfect log phase E. coli RR1ΔM15 cells (Rüther, Nucleic Acids Res10:5765-5772, 1982) after each round of selection (approximately 500times excess of cells compared to eluted phages). After 25 minincubation at 37° C., the cells were centrifuged. The pellet wasdissolved in a small volume of TSB-YE (30 g/l tryptic soy broth, 5 g/lyeast extract) and spread on a TYE plate (15 g/l agar, 10 g/l tryptonewater (Merck), 5 g/l yeast extract, 3 g/l NaCl, 2% glucose and 0.1 g/lampicillin) and thereafter incubated over night at 37° C.

Preparation of Phage Stocks: Phage Infected Cells Grown Over Night onTYE plates were re-suspended in TSB medium (30 g/l tryptic soy broth).An amount of suspended cells corresponding to approximately 100 infectedcells of each eluted phage was inoculated in TSB-YE medium supplementedwith 2% glucose and 100 mg/ml ampicillin. These cells were grown to logphase at 37° C. and a volume of them resembling the same amount of cellsprior to growth were infected with 20 times excess of M13K07 helperphage (New England Biolabs, cat. no. NO315S). Cells and helper phagewere incubated for 30 min at 37° C., and then pelleted bycentrifugation, re-suspended in TSB-YE medium supplemented with 100 mMIPTG (isopropyl-6-D-1-thiogalacto-pyranoside), 25 μg/ml kanamycin and100 μg/ml ampicillin and grown over night at 30° C. An aliquot of there-suspended cells was stored at −20° C. as a glycerol stock.

The induced culture was centrifuged and phages in the supernatant wereprecipitated twice with a PEG/NaCl buffer (20% polyethyleneglycol, 2.5 MNaCl). The phages were re-suspended in selection buffer.

Phage stock and eluted phage were titrated after each round ofselection.

ELISA Analysis of Binding

Proteins from clones obtained after four or five rounds of selectionwere produced in 96-well plates and screened for target binding activityusing an ELISA setup.

Proteins were produced by inoculating single colonies in 1 ml TSB-YEmedium supplemented with 100 μg/ml ampicillin and 1 mM IPTG in deep-wellplates (Nunc, cat. no. 278752) and grown for 18-24 h at 37° C. A smallamount of each culture was transferred to 96-well plates (Costar, cat.no. 9018) and stored at −20° C. as glycerol stocks. Remaining cells werepelleted by centrifugation, re-suspended in 400 μl PBS-T0.05 (PBS+0.05Tween20) and frozen at −80° C. to release the periplasmic fraction ofthe cells. Frozen samples were thawed in a water bath and cells werepelleted by centrifugation. Supernatants containing soluble candidateIgG Fc-binding molecules fused to the albumin binding domain ABD fromStreptococcus strain G148 were assayed for binding in an ELISA asfollows.

Microtiter wells were coated with 100 μl of HSA at 6 μg/ml (Sigma, cat.no. J-1010) in coating buffer (0.1 M sodium carbonate, pH 9.5). Thewells were blocked with 200 μl PBS-T0.05 complemented with 2% 0 driedmilk for 1 h at room temperature. After removal of blocking, 100 ml ofcandidate IgG Fc-binding molecule solution was added in each well andthe plates were incubated for 1.5 h at room temperature. BiotinylatedIgG1κ (at a concentration of 0.05 and 0.5 μg/ml for clones derived fromround 4 and 0.01 μg/ml for clones from round 5) or IgG Fc (at aconcentration of 0.5 μg/ml; Jackson Immunoresearch, cat. no. 009-008,lot 66321) in 100 μl PBS-T0.05 was added to the wells and incubated for1.5 h. Bound target was detected with SA-HRP (Dako, cat. no. P0397),diluted 1:5000 in PBS-T0.05, and incubated for 1 h at room temperature.Plates were washed four times with PBS-T0.05 before incubation with thebiotinylated target, SA-HRP and developing solution. Developing solutionwas prepared by mixing of equal volumes of ImmunoPure TMB kit substratesTMB and H₂O₂ (Pierce, cat. no. 34021), and 100 μl were added to eachwell. After 30 min incubation in darkness, 100 μl stop solution (2 MH₂SO₄) was added. The plates were read at 450 nm in an ELISAspectrophotometer. All steps from blocking to reading were performed ina Tecan Genesis Freedom 200 robot.

Three controls were used:

Well F12: Positive control treated as above, but for plates with clonesfrom selection round 4, a mixture of Z00000 (SEQ ID NO:10) and Z01730(SEQ ID NO:11) as periplasmic fractions was used. For plates with clonesfrom selection round 5, periplasmic fraction of Z00000 was used.

Well G12: Positive control. As described for well F12 but withbiotinylated IgG1κ at a concentration of 1 μg/ml after round 4 and 0.5μg/ml after round 5.

Well H12: Blank. PBS-T0.05 used instead of periplasmic fractions.

Sequencing of Potential Binders

Based on the ELISA results, clones were chosen for sequencing. Forclones taken from selection round 4, clones with absorbance valuessimilar to the positive control (well F12) were given priority. Forclones taken from selection round 5, clones with the highest absorbancevalues were given priority. A high diversity among picked clones wasdesirable, and therefore many clones with different absorbance valueswere chosen from both screens.

PCR fragments were amplified from the chosen colonies using theoligonucleotides AFFI-21 (5′-tgcttccggctcgtatgttgtgtg-3′) and AFFI-22(5′-cggaaccagagccaccaccgg-3′). Sequencing of amplified fragments wasperformed using BigDye® Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, cat. no. 4336919) and the biotinylated oligonucleotideAFFI-72 (5′-biotin-cggaaccagagccaccaccgg-3′) according to themanufacturer's recommendations. The sequencing reactions were purifiedby binding to magnetic streptavidin-coated beads (Magnetic Biosolutions,cat. no. 11103) using a Magnatrix 8000 (Magnetic Biosolutions), andanalyzed on ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Thesequencing results were imported and analyzed with Nautilus software(Thermo Electronics Corporation).

Results Selection

Four different selection setups were applied and five selection roundswere made for each selection setup. Increasing numbers of washes andelution at different pH values were used in the selection setups.

ELISA

Clones obtained after four and five rounds of selection were produced in96-well plates and screened for target binding activity using an ELISAsetup. The putative IgG Fc-binding molecules were in periplasmicfractions obtained by freeze thawing.

In the ELISA screening of clones derived from the fourth selectionrounds, IgG1κ and IgG Fc were used as targets at concentrations of 0.05μg/ml and 0.5 μg/ml for IgG1κ and 0.5 μg/ml for IgG Fc. The ELISAresults for round 4 clones indicated that absorbance values correspondedwell between IgG1 and IgG Fc and were very high even at the lowconcentration of 0.05 μg/ml IgG1.

In the ELISA screening of clones derived from the fifth selectionrounds, IgG1κ was used as target at a concentration of 0.01 μg/ml. Thenumber of background binders was much higher among clones derived fromround 5 as compared to clones from round 4. The responses were lowestamong clones from the IgG_(—)21 selection eluted with pH 4.5.

Sequencing

Clones from round 4 and 5 were sequenced, and the results compared withpreviously known protein Z variants. For the purposes of the presentinvention, one clone derived from round 4 (designated Z02674, SEQ IDNO:7; see FIG. 1) and two clones derived from round 5 (designated Z02726and Z02742, SEQ ID NO:8 and SEQ ID NO:9, respectively; see FIG. 1) werechosen for further characterization.

In summary, the selections from the library ZLib2007-IgG were successfuland suitable candidates were chosen for further characterization.

EXAMPLE 2 Further Characterization of IgG Fc-Binding Polypeptides

In this Example, a group of IgG Fc-binding polypeptides from theselection described in Example 1 were subcloned and expressed inmonomeric form, and their binding characteristics studied.

Materials and Methods Cultivation and Purification

IgG Fc-binding polypeptides Z02674, Z02726 and Z02742, as well as amodified version of Z02674 denoted Z02829, were sub-cloned as monomersinto an expression vector in which expression is regulated by a T7promoter. The IgG Fc-binding polypeptides were expressed with theadditional N-terminal amino acid sequence GSSLQ and the additionalC-terminal amino acid sequence VD. Thus, the expressed Z02674, Z02726and Z02742 molecules have the sequence GSSLQ-[SEQ ID NO:#]-VD, wherein #corresponds to 7, 8 or 9 (see FIG. 1).

E. coli BL21(DE3) cells (Novagen) were transformed with the plasmids andcultivated at 37° C. in 1 l of TSB+YE medium (tryptic soy broth withyeast extract) supplemented with 50 μg/ml kanamycin. At OD₆₀₀=1, IPTGwas added to induce protein expression at a final concentration of 1 mMand the cultivation was incubated at 37° C. for another 5 hours. Thecells were harvested by centrifugation, re-suspended in 200 ml ofbinding buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.0) andsonicated to release the expressed protein. Cell debris was removed bycentrifugation and the supernatant was applied on 40 ml IgG-sepharose inan XK26 column (GE Healthcare). Contaminants were washed away withbinding buffer followed by elution of IgG Fc-binding molecules withelution buffer (0.1 M HAc). The purified IgG Fc-binding molecules weretransferred to 10 mM NH₄HCO₃ by gel filtration and thereafterlyophilized. Concentration was determined using absorption at 280 nm andthe extinction coefficient of the respective protein. The purity of thefinal product was analyzed on SDS PAGE stained with Coomassie Blue. Theidentity of the purified IgG Fc-binding molecules was confirmed usingHPLC-MS.

Solubility Analysis

Lyophilized protein was dissolved in PBS. Protein solution wastransferred to a plastic cuvette and examined for undissolved protein byvisual inspection.

Circular Dichroism Analysis

CD analysis was performed with 0.5 mg/ml protein in PBS. A spectrummeasurement at 195-250 nm was performed at 20° C. The melting point (Tm)of the purified proteins was determined by a variable temperaturemeasurement (VTM) where 220 nm was monitored during heating of thesample to 90° C. After re-equilibrating the sample to 20° C., a newspectrum was taken. An overlay of spectrums before and after VTM showedif the structure was regained after heating to 90° C.

Binding Analysis

Binding of the purified molecules to human IgG was analyzed usingsurface plasmon resonance on a Biacore 2000 instrument (GE Healthcare).Etanercept (trade name Enbrel®, a fusion protein containing the Fcregion of human IgG; Apoteket article no. 566661) and two humanmonoclonal IgG antibodies, palivizumab (trade name Synagis®, does notcomprise a VH3 domain; Apoteket article no. 549170) and trastuzumab(trade name Herceptin®, comprises a VH3 domain: Apoteket article no.573477) were used as target proteins. Target proteins were immobilizedin different flow cells by amine coupling onto the carboxylated dextranlayer on surfaces of CM-5 chips according to the manufacturer'srecommendations. To analyze their binding to the immobilized targetproteins, the purified IgG Fc-binding molecules were diluted in HBS-EP(0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.2)and injected at 25 nM and 100 nM at a constant flow-rate of 25 μl/minfor 4 minutes. The surfaces were regenerated with an injection of 0.3 MHAc, pH 3.2. An estimate of the dissociation equilibrium constant(K_(D)) was made using BIAevaluation 4.1 (GE Healthcare), assuming aone-to-one Langmuir binding model and taking mass transfer effects intoaccount.

Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed to check foraggregates. The purified IgG Fc-binding molecules were diluted to 0.5mg/ml in PBS and 50 μl was injected at the flow rate 0.5 ml/minute on aSuperdex 75 10/300 GL column (GE Healthcare) equilibrated with PBS.

Results Cultivation and Purification

Monomeric IgG Fc-binding molecules were expressed from plasmid vectorsin E. coli. The total amount of IgG sepharose-purified protein from 1liter-cultivations was determined spectrophotometrically at A₂₈₀ nm andis given in Table 2.

TABLE 2 Characteristics and amounts of purified proteins Molecularweight 1 A₂₈₀ = Isoelectric Total amount Protein (Da) (mg/ml) point (mg)Z02674 7321.1 1.05 6.5 30 Z02726 7321.2 1.05 7.7 nd Z02742 7341.2 1.0510.3 60

Lyophilized proteins were dissolved in PBS and 20 μg was analyzed withSDS-PAGE. All protein preparations contained IgG Fc-binding moleculestogether with some contaminating proteins. The size of the IgGFc-binding molecules was confirmed with HPLC-MS.

Solubility Analysis

PBS was added to the lyophilized IgG Fc-binding molecules. Expectedconcentration based on the amount of protein in each vial is shown inTable 3.

TABLE 3 Expected concentration of dissolved molecules. ProteinConcentration (mg/ml) Z02674 18 Z02726 30 Z02742 30

All three protein preparations contained precipitated contaminatingmaterial. For Z02674, undissolved material was removed by centrifugationand the supernatant was kept at +4° C. over night. A new visualinspection was performed, and no new precipitation could be seen. Theconcentration was measured with A₂₈₀ after centrifugation and found tobe 17.4 mg/ml. For Z02726 and Z02742, the pH of the solutions was raisedto approximately 10 with 50% NaOH, which resulted in a clear solutionfor both proteins.

Circular Dichroism Analysis

CD analysis was performed with the proteins Z02726, Z02742 and Z02829.Z02829 is a derivative of Z02674 containing two substitutions at thebeginning of the protein (A8N and W11N with respect to the sequence ofthe entire expressed molecule, i.e. A3N and W6N with respect to SEQ IDNO:7). Thus, the IgG Fc-binding motif of Z02829 is the same as that ofZ02674.

The determined melting points of the IgG Fc-binding molecules are givenin Table 4.

TABLE 4 Determined melting points. Protein T_(m) (° C.) Z02829 62 Z0272662 Z02742 63

Overlay of spectra taken at 195-250 nm before and after VTM are shown inFIG. 2A-2C. As is evident from these overlay diagrams, all three IgGFc-binding polypeptides completely regained their structure afterheating at 90° C.

Binding Analysis

Binding of the purified polypeptides to human IgG Fc was analyzed usingsurface plasmon resonance on a Biacore 2000 instrument. Palivizumab(without VH3 domain), trastuzumab (with VH3 domain) and etanercept(TNFαr-Fc fusion) were immobilized on chip surfaces with amine-coupling.The Z protein Z00000 (SEQ ID NO:10) and the earlier obtained variantZ01730 (SEQ ID NO:11) were used as controls. Binding diagrams for IgGFc-binding molecules injected at 25 nM over immobilized target proteinsare shown in FIGS. 3A-3C and FIGS. 4A-4C.

As evidenced in these Figures, all tested polypeptides bind to IgG.

In order to calculate an estimated binding affinity, the diagramsobtained from binding to palivizumab were analyzed with theBIAevaluation software provided by the manufacturer. The results arepresented in Table 5. As shown in this Table, the IgG Fc-bindingpolypeptides exhibit binding affinities for IgG which are comparable tothe positive control Z00000, which is a well known IgG Fc-bindingmolecule.

TABLE 5 Binding constants for selected IgG Fc-binding molecules Proteink_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) Z02674 2.4 × 10⁶ 5.2 × 10⁻² 22Z02726 2.6 × 10⁶ 4.5 × 10⁻² 18 Z02742 9.6 × 10⁶ 4.3 × 10⁻² 5 Z00000(control) 3.6 × 10⁵ 4.9 × 10⁻³ 14

Size Exclusion Chromatography

SEC analysis was performed by injecting 100 μg of purified protein on aSuperdex 75 10/300 GL column equilibrated with PBS. All IgG Fc-bindingmolecules eluted in single peaks. The shape of peaks differed betweenthe IgG Fc-binding molecules and they elute at different times. However,they all elute later than Z00000, which indicates that there are noaggregates.

EXAMPLE 3 Affinity Chromatography Study of Elution pH and Capacity ofIgG Fc-Binding Polypeptides

In this Example, individual IgG Fc-binding polypeptides from theselection described in Example 1 were coupled to chromatoghraphic media,and their elution conditions and binding capacities were studied inaffinity chromatography experiments.

Materials and Methods Immobilization of IgG Fc-Binding Polypeptides

The inventive IgG Fc-binding polypeptides Z02742, Z02674 and Z02726 andthe reference molecule Z00000 were each immobilized on NHS-activatedHiTrap™ columns (0.962 ml, GE Healthcare). The immobilization, ligandcoupling via primary amines, was performed in accordance with themanufacturer's instructions. Each polypeptide was immobilized on fourcolumns, of which two were used for the elution pH study and two wereused for the capacity study.

Buffer Preparation

Citric acid and NaCl (Merck) were dissolved in water to finalconcentrations of 0.1 M and 0.9 percent by weight (% wt/wt)respectively. Two buffers were prepared from this solution by adjustingpH to 6.2 for one part of the solution (buffer A) and to 2.5 for theother part of the solution (buffer B). pH adjustments were made byaddition of NaOH. The buffers were filtered prior to use.

Elution Study

Elution pH was studied for three different samples run on columnscomprising IgG Fc-binding polypeptide ligands. The samples weretrastuzumab (trade name Herceptin®, Apoteket article no. 573477),etanercept (trade name Enbrel®, Apoteket article no. 566661) andpalivizumab (trade name Synagis®, Apoteket article no. 549113). Thesamples were prepared according to the manufacturer's instructions andwere thereafter diluted to 1 mg/ml solutions in buffer A.

The columns were attached to an ÄKTA™ explorer 10 S chromatographysystem (GE Healthcare) and equilibrated (4 column volumes (CV) buffer A,flow rate 1 ml/min). Sample solution was injected into a Superloop™ (50ml, GE Healthcare) and 2 ml were loaded on each column at a flow rate of0.4 ml/min. The columns were washed (3 CV buffer A, 1 ml/min) and thesample was eluted by an acidic pH gradient (25 CV, 0-100% buffer B, 1ml/min). After the acidic pH gradient, the columns were washed (4 CV100% buffer B, 1 ml/min) and re-equilibrated (4 CV buffer A, 1 ml/min).

Eluted samples were collected with a fraction collector (Frac-950, GEHealthcare) in order to allow pH measurements in the eluted fractions.Peaks were collected when the absorbance at 280 nm (A₂₈₀) exceeded 5% ofA₂₈₀ for the 1 mg/ml sample solution. Peak collection stopped when theabsorbance fell below the same 5% threshold.

Capacity Study

Dynamic binding capacity for a chromatographic medium is usually definedas the amount of sample applied to the medium when the absorbance at 280nm reaches 10% of the sample absorbance at 280 nm. The capacity wasdetermined by loading sample on columns comprising immobilized IgGFc-binding polypeptides. To determine the capacity, the dead volume(i.e. tubing and column volume) was subtracted from the sample volumerequired for 10% breakthrough. The dead volume was measured by runningsample through a column comprising no IgG Fc-binding polypeptide.

Human polyclonal IgG (trade name Gammanorm®, Apoteket article no.096169, comprising a mix of VH3 subfamily and non-VH3 subfamilyantibodies) was used for determining capacity. The IgG sample wasprepared by diluting 165 mg/ml Gammanorm® to 0.75 mg/ml with 1×PBS.

The columns were attached to an ÄKTA™ explorer 10 S chromatographysystem (GE Healthcare) and equilibrated (4 CV buffer A, 1 ml/min). Thesample was loaded on the columns with a flow rate of 0.241 ml/min(residence time 4 min) until A₂₈₀ reached 10% (in this case 128.2 mAU)of the sample absorbance. Bound protein was eluted (10 CV buffer B, 1ml/min) and the columns were re-equilibrated (4 CV buffer A, 1 ml/min).

Results Elution Study

All samples were eluted at a higher pH (i.e. earlier in the gradient)from the columns comprising immobilized Z02674 than from the columnscomprising the other polypeptide ligands. Trastuzumab was eluted fromcolumns comprising Z00000 at a lower pH (i.e. later in the gradient)than from the other columns. Thus, pH in eluted fractions of trastuzumabwere higher from the columns comprising the IgG Fc-binding polypeptidesaccording to the invention than from the columns comprising thereference molecule Z00000. Overlays of chromatograms for differentcolumns are shown in FIGS. 5A-C.

Thus, the tested IgG Fc-binding polypeptides according to the inventionbind to IgG and exhibit elution profiles in affinity chromatographywhich are comparable to, or better than, those of the column-coupledreference molecule Z00000.

Capacity Study

Capacities of the column-coupled IgG Fc binding polypeptides accordingto the invention ranged from 0.23 to 0.33 moles of IgG/moles polypeptideligand and were comparable with the capacities of column-coupled Z00000(see FIG. 6). The dynamic binding capacity of columns comprisingimmobilized Z02674 was, however, approximately 20-30% higher than forthe other columns.

1. Immunoglobulin G Fc region binding polypeptide, comprising animmunoglobulin G Fc region binding motif (BM) which motif consists of anamino acid sequence selected from:i) EQQX₄AFYEIL HLPN LTEX₁₈QX₂₀ X₂₁AFIX₂₅X₂₆LRX₂₉,

wherein, independently of each other, X₄ is selected from H and N; X₈ isselected from D and G; X₂₀ is selected from R and K; X₂₁ is selectedfrom H and Q; X₂₅ is selected from R, A and G; X₂₆ is selected from A, Sand T; and X₂₉ is selected from G, K and A; and ii) an amino acidsequence which has at least 85% identity to the sequence defined in i).2. IgG Fc-binding polypeptide according to claim 1, wherein X₄ is H. 3.IgG Fc-binding polypeptide according to claim 1, wherein X₁₈ is G. 4.IgG Fc-binding polypeptide according to claim 1, wherein X₂₀ is K. 5.IgG Fc-binding polypeptide according to claim 1, wherein X₂₁ is H. 6.IgG Fc-binding polypeptide according to claim 1, wherein X₂₅ is R. 7.IgG Fc-binding polypeptide according to claim 1, wherein X₂₆ is A. 8.IgG Fc-binding polypeptide according to claim 1, wherein X₂₉ is G. 9.IgG Fc-binding polypeptide according to claim 1, wherein the amino acidsequence i) is selected from one of SEQ ID NO:1, SEQ ID NO. 2, and SEQID NO.
 3. 10. IgG Fc-binding polypeptide according to claim 9, whereinthe amino acid sequence is SEQ ID NO:1.
 11. IgG Fc-binding polypeptideaccording to claim 1, in which said IgG Fc-binding motif forms part of athree-helix bundle protein domain.
 12. IgG Fc-binding polypeptideaccording to claim 11, in which said IgG Fc-binding motif essentiallyforms part of two alpha helices and a loop connecting them, within saidthree-helix bundle protein domain.
 13. IgG Fc-binding polypeptideaccording to claim 12, in which said three-helix bundle protein domainis selected from domains of bacterial receptor proteins.
 14. IgGFc-binding polypeptide according to claim 13, in which said three-helixbundle protein domain is selected from domains of protein A fromStaphylococcus aureus or derivatives thereof.
 15. IgG Fc-bindingpolypeptide according to claim 14, which comprises an amino acidsequence selected from: ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK,ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK;AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; andVDNKFNK[BM]-DPSQSANLLAEAKKLNDAQAPK;

wherein BM is an IgG Fc-binding motif wherein the motif consists of anamino acid sequence selected from: i) EQQX₄AFYEIL HLPNLTEX₁₈QX₂₀X₂₁AFIX₂₅X₂₆LRX₂₉, wherein, independently of each other, X₄ isselected from H and N; X₈ is selected from D and G; X₂₀ is selected fromR and K; X₂₁ is selected from H and Q; X₂₅ is selected from R, A and G;X₂₆ is selected from A, S and T; and X₂₉ is selected from G, K and A;and ii) an amino acid sequence which has at least 85% identity to thesequence defined in i).
 16. IgG Fc-binding polypeptide according toclaim 12, which comprises the amino acid sequence:FWK-[BM]-IDPSQSARLLAXaAKKLDDAQ

wherein: BM is an IgG Fc-binding motif wherein the motif consists of anamino acid sequence selected from: i) EQQX₄AFYEIL HLPNLTEX₁₈QX₂₀X₂₁AFIX₂₅X₂₅X₂₆LRX₂₉, wherein independently of each other X₄is selected from H and N; X₈ is selected from D and G; X₂₀ is selectedfrom R and K; X₂₁ is selected from H and Q; X₂₅ is selected from R, Aand G: X₂₆ is selected from A, S and T; and X₂₉ is selected from G, Kand A; and ii) an amino acid sequence which has at least 85% identity tothe sequence defined in i); and X_(a) is selected from R, G and Q. 17.IgG Fc-binding polypeptide according to claim 12, which comprises theamino acid sequence: VDAKFWK-[BM]-DPSQSARLLAX_(a)AKKLDDAQAPK

wherein: BM is an IgG Fc-binding motif wherein the motif consists of anamino acid sequence selected from: i)EQQX₄AFYEIL HLPN LTEX₁₈QX₂₀AFIX₂₅X₂₆RX₂₉,

wherein, independently of each other, X₄ is selected from H and N; X₈ isselected from D and a X₂₀ is selected from R and K; X₂₁ is selected fromH and Q; X₂₆ is selected from R, A and X₂₆ is selected from A, S and T;and X₂₉ is selected from G, K and A; and ii) an amino acid sequencewhich has at least 85% identity to the sequence defined in i); and X_(a)is selected from R, G and Q.
 18. IgG Fc-binding polypeptide according toclaim 16, wherein Xa is R.
 19. IgG Fc-binding polypeptide according toclaim 16, which comprises an amino acid sequence selected from one ofSEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:
 6. 20. IgG Fc-bindingpolypeptide according to claim 19, which comprises the amino acidsequence SEQ ID NO:4.
 21. IgG Fc-binding polypeptide, comprising anamino acid sequence which comprises a sequence which fulfils onedefinition selected from the following: it is selected from SEQ IDNO:7-9; and it is an amino acid sequence having 85% or greater identityto a sequence selected from SEQ ID NO:7-9.
 22. IgG Fc-bindingpolypeptide according to claim 21, wherein the amino acid sequencecomprises a sequence which fulfils one definition selected from thefollowing: it is SEQ ID NO:7; and it is an amino acid sequence having85% or greater identity to SEQ ID NO:7.
 23. IgG Fc-binding polypeptideaccording to claim 21, comprising additional amino acid residues Cterminally and/or N terminally with respect to said IgG Fc-bindingpolypeptide.
 24. IgG Fc-binding polypeptide according to claim 23, inwhich each amino acid extension enhances binding of IgG Fc by thepolypeptide.
 25. IgG Fc-binding polypeptide according to claim 23, inwhich the or each amino acid extension improves production,purification, stabilization in vivo or in vitro, coupling, or detectionof the polypeptide.
 26. IgG Fc-binding polypeptide according to claim21, which binds to IgG Fc such that the K_(D) value of the interactionis at most 1×10⁻⁶ M.
 27. IgG Fc-binding polypeptide according to claim26, which binds to IgG Fc such that the K_(D) value of the interactionis at most 1×10⁻⁷ M.
 28. IgG Fc-binding polypeptide according to claim27, which binds to IgG Fc such that the K_(D) value of the interactionis at most 5×10⁻⁸ M.
 29. IgG Fc-binding polypeptide according to claim 1or 21, which is capable of binding to the Fc portion of a human IgGmolecule.
 30. IgG Fc-binding polypeptide according claim 29, which iscapable of binding to classes 1, 2 and 4 of human IgG.
 31. IgGFc-binding polypeptide according to claim 1 or 21, which is capable ofbinding to the interface between the CH2 and CH3 domains of IgG Fc. 32.IgG Fc-binding polypeptide according to claim 1 or 21, which is capableof binding to an area on the Fc molecular surface made up by the Fcamino acid residues T250-S254, T256, L309-L312, L314, D315, E430 andL432-Y436.
 33. IgG Fc-binding polypeptide according to claim 1 or 21 inmulti meric form, comprising at least two IgG Fc-binding polypeptidemonomer units, whose amino acid sequences may be the same or different.34. IgG Fc-binding polypeptide according to claim 33, in which the IgGFc-binding polypeptide monomer units are covalently coupled together.35. IgG Fc-binding polypeptide according to claim 33, in which the IgGFc-binding polypeptide monomer units are expressed as a fusion protein.36. A polynucleotide encoding a polypeptide according to claim 1 or 21.37. Method of producing a polypeptide according to claim 1 or 21,comprising expressing a polynucleotide encoding a polypeptide. 38.Method of isolating molecules comprising IgG Fc from a sample, whichcomprising the steps: (i) providing a sample containing moleculescomprising IgG Fc; (ii) contacting the sample with an IgG Fc-bindingpolypeptide according to claim 1 or 21, whereby said moleculescomprising IgG Fc bind to the polypeptide; (iii) isolating boundmolecules comprising IgG Fc from the sample.
 39. Method according toclaim 38, in which said sample is derived from cells expressingmolecules comprising IgG Fc.
 40. Method according to claim 38, in whichsaid molecules comprising IgG Fc are IgG molecules or fragments thereof.41. Method according to claim 40, in which said IgG is human IgG. 42.Method according to claim 40, in which said IgG are monoclonal IgGantibodies.
 43. Method according to claim 38, in which said moleculescomprising IgG Fc are Fc fusion proteins.
 44. Method according to claim38, in which said IgG Fc-binding polypeptide is immobilized on achromatography matrix.
 45. Method of producing molecules comprising IgGFc, which method comprises the steps: (i) expressing desired moleculescomprising IgG Fc; (ii) obtaining a sample of molecules comprising IgGFc from said expression; (iii) contacting the sample with an IgGFc-binding polypeptide according to claim 1 or 21, whereby moleculescomprising IgG Fc bind to the polypeptide; (iv) isolating boundmolecules comprising IgG Fc from the sample, and (v) recovering boundmolecules comprising IgG Fc through elution thereof from the IgGFc-binding polypeptide.
 46. Method according to claim 45, in which saidmolecules comprising IgG Fc are IgG molecules or fragments thereof. 47.Method according to claim 46, in which said IgG is human IgG.
 48. Methodaccording to claim 46, in which said IgG are monoclonal IgG antibodies.49. Method according to claim 45, in which said molecules comprising IgGFc are Fc fusion proteins.
 50. Method according to claim 45, in whichsaid IgG Fc-binding polypeptide is immobilized on a chromatographymedium.
 51. Affinity chromatography medium, comprising an IgG Fc-bindingpolypeptide according to claim 1 or
 21. 52. IgG Fc-binding polypeptideaccording to claim 17, wherein Xa is R.
 53. IgG Fc-binding polypeptideaccording to claim 34, in which the IgG Fc-binding polypeptide monomerunits are expressed as a fusion protein.